1. Field of the Invention
The present invention relates to substances of lipo-oligosaccharide structure which are capable of forming plant-specific symbiotic signals, to processes for producing these substances and to their applications.
2. Discussion of the Background
As it is known, plants require for their growth a source of combined nitrogen such as ammonia or nitrate. The fixing of nitrogen, by chemical or biological reduction of atmospheric nitrogen (N.sub.2) to ammonia (NH.sub.3), therefore plays a vital role in agricultural production.
As it is also known, symbiotic microorganisms can promote the growth and development of plants through biological fixation of nitrogen.
Rhizobiaceae are Gram negative soil bacteria which generally fix nitrogen in symbiotic association with plants: the establishment of such symbiosis with nitrogen-fixing bacteria allows numerous plant species to grow in soils with low assimilable nitrogen levels. By virtue of photosynthesis, the plant partner provides the bacteria with the energy required for reducing molecular nitrogen to ammonia. In return, the ammonia fixed by the microsymbiont is provided to the host plant which incorporates it into its nitrogen metabolism. The symbiotic association which is established between nitrogen-fixing bacteria such as Rhizobiaceae and plants of the Leguminoseae family is the most important from an ecological and agronomic point of view. This association leads to the formation of nodosities or nodules mainly on the roots of the host plants. Inside these nodosities, the bacteria reduce atmospheric nitrogen to ammonia by means of the nitrogenase enzymatic complex.
The symbiosis between nitrogen-fixing bacteria of the Rhizobiaceae family (Rhizobium, Bradyrhizobium, Sinorhizobium and Azorhizobium genera ) and plants of the Leguminoseae family therefore play a very important role in temporate and tropical agriculture. Oil- and protein-rich plants such as soybean and groundnut, fodder plants such as lucerne and clover, protein-rich plants such as peas and field bean, food plants such as beans, peas, lentils and chickpeas, green manures such as Sesbania and the like. By virtue of these symbioses, the cultivation of Leguminoseae is often less costly in nitrogenous fertilizer than the cultivation of plants belonging to other families. In this respect, it should be noted that the massive use of nitrogenous fertilisers has certain disadvantages. Firstly, the synthesis, transportation and application of fertilizers is costly in fossil energy, and this has several consequences: at the farming level it increases the production hosts of the farmers, and at the environmental level it contributes to the greenhouse effect by increasing the CO.sub.2 content. Moreover, an ill thought-out or excessive application of nitrogenous fertilizers causes pollution of fresh waters with eutrophication of the surface waters and an increase in the nitrate content of the ground water table. These various reasons militate in favor of an increased use of biological fixation of nitrogen.
Given the very damaging consequences of the excessive use of nitrogenous fertilizers, it is therefore necessary to increase the contribution of the biological fixation of nitrogen by plants and in particular by the cultivated species which play an important role in human and animal nutrition. The most acceptable solution both from the ecological and economical point of view is to improve the Rhizobiaceae-Leguminoseae symbiosis.
It has been proposed to carry out this improvement by providing Rhizobiaceae (in particular Rhizobium or Bradyrhizobium) at the time of sowing, either by coating the seeds or by means of granules mixed with the seeds or by means of cultivation in liquid medium. These bacteria supplies are however effective only in the relatively rare cases where the appropriate symbiotic bacteria are naturally absent or are not very abundant in the soils. In the opposite cases, that is to say in soils already containing these bacteria, it is practically impossible to impose a strain which is deliberately introduced, due to competition with the indigenous bacteria present in the soils, which, even if they are not necessarily effective for fixing nitrogen, constitute nevertheless a limiting factor for introducing selected bacteria.
It has also been proposed to enhance the Rhizobium-Leguminoseae symbiosis by treating the plants with an exopolysaccharide derived from bacteria of the Rhizobium genus or with an oligosaccharide containing one or more units of such an exopolysaccharide (EPS)--cf. the PCT International Application published under the No. WO 87/06796 filed on behalf of THE AUSTRALIAN NATIONAL UNIVERSITY and mentioning as inventors:. B. G. ROLFE, S. P. DJORDJEVIC, J. W. REDMOND and M. BATLEY. However, this EPS is a product encoded by non-symbiotic genes. In fact, the biosynthesis of these exopoly-saccharides is not under the direct control of the nod genes which control infection and nodulation. These exopolysaccharides are synthesized by Rhizobium strains whose plasmid pSym has been cured, which plasmid carries most of the symbiotic genes and in particular the nod genes.
The genes involved in the nodule-formation process have been localized and several common and specific nodulation genes (nod genes) have been identified and characterized (see Long, S. R., Cell, 1989, 56, 203-214). Whereas the nodA,B,C genes are nodulation genes which are common to the various species of symbiotic Rhizobiaceae, specific nod genes exist which determine the host spectrum and which therefore vary in the various species, and regulatory genes of the nodD type which control the expression of the entire nod genes.
The common genes nodA,B,C have been identified in the four bacterial genera which are capable of establishing a nitrogen-fixing symbiosis with the Leguminoseae: Rhizobium, Bradyrhizobium, Sinorhizobium and Azorhizobium. For the Rhizobium genus, the nucleotide sequeuence of these genes has been obtained in R. meliloti (Torok et al., Nucleic Acids Res., 1984, 12, 9509-9522; Jacobs et al., J. Bacteriol., 1985, 162, 469-476; Egelhoff et al., DNA, 1985, 4, 241-242). R leguminosarum (Rossen et al., Nucl. Acids Res., 1984, 12, 9497-9508 ), R. trifolii (Schofield et al., Nucl. Acids Res:., 1986, 14, 2891-2903).
For Bradyrhizobium sp. ( Scott, Nucl. Acids Res., 1986, 14, 2905-2919).
For Azorhizobium caulinodans (Goethals et al., Mol. Gon. Genet., 1989, 219, 289-298).
A team of researchers comprising several of the inventors of the present invention have shown that in Rhizobium meliloti, the common genes nodA,B,C induce, conjointly with the specific genes nodH and nodQ, the production of the host-specific extracellular Nod signals present in the culture supernatants of these bacteria: cf. FAUCHER et al., J. BACTERIOL (1988), 172, 5489-5429 and FAUCHER et al., Molec. Plant-Microbe Interact. (1989), 2, 291-300, among others. Furthermore, the latter of these two publications gives an account of the fractionation of the sterile supernatant by ultrafiltration thus making it possible to reveal the presence of two Nod factors with an apparent molecular mass of less than 5,000 Da.
The specificity of infection and nodulation is determined in R. meliloti at two levels, namely:--the nodD genes activate the expression of other nod operons depending on the presence of specific signals produced by the plants (Gyorgypal et al., Molec. Gen. Genet., 1988, 212, 85-92) and, --specific genes such as nodH and nodQ determine, when they are activated, the production of bacterial extracellular signals (Nod factors) which make it possible to recognize and stimulate a host leguminous plant such as lucerne (Faucher et al., J. Bacteriol, 1988, 172, 5489-5499; Faucher et al., Molec. Plant-Microbe Interact., 1989, 2, 291-300 ) . However, the chemical structure of the bacterial signals was not known.